JPS59154994A - Preparation of amino acid by fermentation - Google Patents

Abstract

PURPOSE:To prepare an amino acid in high efficiency, by culturing a microbial strain capable of producing amino acid and belonging to a glutamic acid-producing coryne-type bacterium. CONSTITUTION:A microbial strain belonging to glutamic acid-producing coryne- type bacterium capable of producing amino acid, and having promoted activity to proliferate using L-aspartic acid as a nitrogen source, e.g. RL-9-14 strain (FERM-P No.6914) derived from Corynebacterium glutamicum ATCC21543, is cultured in a nutrient medium containing L-aspartic acid under aerobic condition at 24-37 deg.C and 5-9pH for 2-7 days, and amino acids such as L-lysine, L-threonine, L-isoleucine, L-arginine, etc. are collected from the cultured liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing amino acids by fermentation. More specifically, the present invention is directed to culturing amino acid-producing microorganisms, which belong to coryneform glutamate-producing bacteria and have an enhanced ability to grow using L-aspartic acid as a nitrogen source, in a nutrient medium to accumulate amino acids in the culture solution. The present invention relates to a method for producing amino acids by a fermentation method, which is characterized in that the amino acids are collected from the culture solution. The purpose is to provide an industrially advantageous method for producing amino acids.

Conventionally, various mutant strains have been used in the production of amino acids by fermentation methods, and these have contributed to the economical industrial production of amino acids. However, in recent years, as demand for amino acids for medicine, food, feed, and other applications has increased, improvements in their production methods have been increasingly desired.

In view of this situation, the present inventors have conducted intensive research on improving the amino acid production ability of amino acid-producing bacteria, and as a result, they have discovered that they belong to the coryneform glutamate-producing bacteria and utilize L-aspartic acid as a nitrogen source. We have found that amino acid productivity can be significantly improved by using microorganisms with enhanced ability to grow. This phenomenon of improved amino acid productivity is particularly noticeable when L-aspartic acid or a salt thereof is added as a fermentation substrate to the fermentation medium together with other carbon sources or nitrogen sources.

The reason why microorganisms with such properties have excellent amino acid productivity is unknown, but the increase in cell membrane permeability of L-aspartic acid or the enhancement of the metabolic pathway of L-aspartic acid may result in the production of amino acids. It is thought that this may lead to an enhancement of the activity of the enzymes produced.

Conventionally, there has been no known method for producing amino acids using microorganisms belonging to coryneform glutamate-producing bacteria that have enhanced growth ability using L-aspartic acid as a nitrogen source, and the present invention is based on this new knowledge. It was completed by

The present invention will be explained in detail below.

The microorganism used in the present invention is a microorganism that has amino acid productivity and has an enhanced ability to grow using L-aspartic acid as a nitrogen source, by imitating purine-type glutamic acid-producing bacteria.

Coryneform glutamic acid-producing bacteria refers to L-glutamic acid-producing bacteria belonging to the genus Corynebacterium, Brevibacterium, Microbacterium, and the like.

Furthermore, microorganisms having the properties related to the present invention can be selected from among microorganisms that grow faster than the parent strain in a minimal medium containing L-aspartic acid as the only nitrogen source; It can also be obtained by conventional mutation treatments, cell expansion methods, transduction methods, and other genetic techniques. Furthermore, the microorganism may also have other properties (for example, various nutritional auxotrophies, resistance to preservatives, sensitivity to drugs, dependence on preservatives, etc.).

The amino acids produced include L-lysine, L-threonine, L-isoleucine, and L-arginine.

Specific examples of microorganisms used in the present invention include the following.

As an L-lysine producing bacterium, Corynebacterium glutamicum ATCC21543 (homo!=
fCRL-9-14 (Fiber auxotrophy - leucine auxotrophy - penidillin resistance - thiarisine resistance) and Brevibacterium lactofermentum ATCC21759 (pantothenic acid auxotrophy,
@652-20 (Feikoken Bacterial Serial No. 69/S) derived from Corynebacterium glutamicum ATCC216 as an L-threonine producing bacterium.
-440- derived from 60 (methionine requirement - thialidine resistance - α-amino-β-hydroxyvaleric acid resistance)
9 (Feikoken Bacterial Report No. 69/is) and Brevibacterium flavum ATC021269 (α-amino-β
-Hydroxyvaleric acid resistance)
Corynebacterium glutamicum FER as an L-isoleucine producing bacterium.
-39-2x-15 derived from M P-6830 (arginine auxotrophy - thiarisine resistance)
9/8 issue). These strains are L.
- It can be clearly distinguished from the parent strain in that it can grow rapidly using L-aspartic acid as a nitrogen source in a minimal medium containing aspartic acid as the sole nitrogen source.

Next, a specific example of the operation for obtaining such a microorganism will be described below regarding L-inleucine production [1] by the genus Corynebacterium.

Note that other bacteria used in the present invention can also be obtained by the same method.

Operation example Using Corynebacterium glutamicum L-inleucine producing bacterium FERM p-6s3o in a conventional manner j'), N
- Methyl-N+-Nitro-N-nitrosoquanidine treatment (250 μf/m at 130°C for 30 minutes) and then plated on the surface of the minimal agar medium described below containing L-aspartic acid as the sole nitrogen source. 3 at 30℃
It was statically cultured for 1 day. Originally, the parent stock FERM P-6
830 has a weak ability to assimilate L-aspartate, so weak growth of cells of the parent strain was observed on the entire surface of the agar medium, but among them, cells of the parent strain were observed to grow at a frequency of 10-6 to 10-8. The mutant strain was observed to grow quickly and form large colonies. These fast-growing colonies were also treated with L-asparagine [? was plated on a minimal agar medium with C. aspartic acid as the sole nitrogen source, and cultured statically at 30°C for 2 days, and 30 strains that grew significantly faster than the parent strain were obtained using shea aspartic acid as the sole nitrogen source. It was isolated as a microorganism with enhanced growth ability. It was confirmed that all of these strains grew at the same speed as the parent strain on a normal minimal agar medium containing ammonium sulfate as a nitrogen source in place of L-aspartic acid.

These 30 strains were subjected to an L-inleucine fermentation test (Example 1
), and lj39-21-15 was selected as a strain with superior shim-isoleucine production compared to the parent strain.

Composition of minimal medium with L-aspartic acid as the sole nitrogen source Nigelcose o, sy/di %L-7 Sodium spapartate 0.15 f/di %KHzPOa 0.1
5 fl/”', K2HPO40,05f7'dl, N
aCA! 0.01 f/cLi, MfSO4-7Hz
OO, 05f/di, CaC1x ・2H*O'1μ
yAl, MncJ,.

4H207μf/ml, FeSO4-7H2010μy
/mt, Thiamine-H (Vo, 1μf/rn1. Biotin 30μφ, L-Argini 7-HCI 501t
f/fttl, agar 1.5fj/lit.

pH 7.2 (with NaOH) The method for producing the relevant amino acids using these microorganisms is to use a regular Eihin medium containing carbon sources, nitrogen sources, inorganic salts, growth factors, etc. This can be done by law.

Carbon sources used include carbohydrates such as glucose, sucrose, lactose, molasses, starch, and starch hydrolysates, organic acids such as acetic acid, propionic acid, formic acid, lactic acid, pyruvic acid, fumaric acid, and malic acid, and L- Amino acids such as glutamic acid and L-asparagine, methanol,
Alcohols such as ethanol and n-glopanol and other hydrocarbons can be used alone or in combination.

As the nitrogen source, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphorus, urea, aqueous ammonia, and others can be used.

As inorganic salts, potassium dihydrogen phosphate, potassium monohydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, etc. can be used.

Furthermore, in the case of a mutant strain exhibiting auxotrophy, nutrients can be added as pure standards or natural products containing them.

According to the present invention, by containing L-aspartic acid or its salt in the medium, the yield of the target amino acid can be further increased.

The salts of L-aspartic acid are not particularly limited, but include sodium salts, potassium salts, ammonium salts, calcium salts, magnesium salts, amine salts (e.g. ethylenediamine salts), salts with basic amino acids (e.g. ornithine, lysine, etc.). The average of ) * can be used.

The amount of L-aspartic acid or its salt added to the culture medium is required to achieve the purpose in terms of aspartic acid of o, o sy/d1 or more, and if the limit of effectiveness and economic efficiency are taken into account, it should be less than 10 y/d6. It is desirable that there be. Most preferably, it is 0.1 to 3 y/di.

It goes without saying that L-aspartic acid or its salt also serves as a carbon source and a distillate source.

The preferred fermentation conditions for fermentation production of amino acids are aerated culture, fermentation temperature 24-37°C, and fermentation days 2-7.
It is days. The pH of the fermentation liquid is maintained in the range of 5 to 9, but inorganic or organic acids or alkalis, as well as urea, calcium carbonate, ammonia gas, etc. are used to adjust the pH.
Magnesium phosphate and the like can be used.

Isolation of amino acids from fermentation liquor is usually carried out by combining ion exchange resin method and other known methods.

Examples are shown below.

Example 1 L-inleucine producing bacteria of Corynebacterium glutamicum @39-2l-15 (arginine auxotrophy - thiarisine stiffness - L-aspartate assimilation enhanced strain) was grown at 30%
In a 0 d Erlenmeyer flask (7) 2 (Mt/seed medium (glucose s f/'%) yeast extract 1 f/'d, 11 hept 71 W/di, urea 0.3 f/di, NaCJ
O, 25f/LO11%Co-7・Ste4-7'・Lee-)) -o, sy/dl, biof;/50
P1/l, pH 7,2) and incubated at 28°C for 24 hours.
Time, 210 rpm.

Shaking culture was performed on a rotary shaker at a rotation speed of . 2 d of this seed culture solution was inoculated into two types of fermentation medium: 20+114 fermentation medium (1) (described later) and this fermentation medium (1) to which 0.5 f/di of ammonium L-aspartate was added. The cells were cultured for 72 hours in the same manner as the seed culture described above. As a control, the parent strain Corynebacterium glutamicum FERMP-6830 was similarly cultured. As a result, the amount of L-inleucine accumulated was shown in Table 1, and the amount of L-isoleucine accumulated was high in the mutant strain.

Table 1 Composition of fermentation medium (1): Blackstrap molasses (in terms of glucose) 8.
0shihiro, corn steep liquor 〇, 5φ11 ammonium sulfate 2
Shi4, urea 0.3 f/di, KF (2PO40,2
P/dl, K2HPO, 0.05fl/di, MfSO
4-7H200, 05f/di.

FeSO4-7H20o, o 01 f/di, MnC
! , 2-4H200,'001f/d11ZnSO,-
7H201M9/13, Biotia 50 pW/11
.

Arginine hydrochloride 500μ (pH 7,4) Example 2 Corynebacterium glutamicum L-lysine producing strain RL-9-14 (L-lysine-producing strain derived from ATCC 21543) and Brevibacterium lactofermentum L-lysine producing bacterium 652-20 (L derived from ATCC 21759)
- A strain with enhanced aspartate assimilation ability) is used.

These strains were combined with glucose 4 f/di, HoIJ Hair')n 29/+fJ, KM2P0.0.1 s
f/di, x, Hpo40, 05 f/dl, Mf
BO4・7HxOO, 0' 5 f/di, biotin 5
0μv/l, urea o, ay/di, yeast extract 0
．． 5flag (pH 7,2) at 30°C.
The fermentation medium (2) with the following composition and 0.5 ammonium L-aspartate were cultured with shaking for 24 hours.
5% in duplicate fermentation medium of medium supplemented with t/di.
(v/v) and cultured with shaking at 30°C for 4 days.

As controls, parent strains ATCC21543 and ATCC
21759 was used and cultured in the same manner.

As a result, the amount of L-lysine accumulated was as shown in Table 2.

Composition of fermentation medium (2): Molasses (in terms of glucose) 9 f
/di, soybean meal sulfuric acid decomposition product (soybean meal equivalent) 2%l,
KH2PO40,07fy'dt, MfSO,・7H
,00,05-1 urea (J, sy/di, ammonium sulfate 0
5, CaCO32f/dl (pH 7.5, ammonia neutralization) Table 2 Example 3 As the inoculum, Corynebacterium glutamicum L
- Threonine producing bacteria 440-9 (L-aspartate assimilation enhanced strain derived from ATCC 21660) and Brevibacterium flavum L-threonine producing AM31
1-6 (L-aspartate assimilation ability enhanced strain derived from ATCC21269) is used.

These strains were cultured in the same manner as in Example 2, and
As a result of inoculating and culturing two sets of fermentation medium (3) with the following composition and a medium to which 0.5η of L-aspartate ammonium was added, the results are shown in Table 3. Obtained.・Composition of fermentation medium (3) Nigel course 8V
Ammonium sulfate 2 gzg, KH2PO40,05t/di
, K, HPO40,05g/#.

MfSO, 7H200, 1f/di, FeS○,
-7H,OO,001r/#,Mn5o, -4H26
0, o o 1y, m, L-methionine 150 μυ odor, biotin 100 μf/l, CaCO32f/dl
(I) H7, 4) Table 3

Claims (5)

[Claims] (1) Amino acid-producing microorganisms that belong to the coryneform glutamate-producing bacteria and have an enhanced ability to grow using L-aspartic acid (as a primary source) are cultured in a nutrient medium, and amino acids are added to the culture solution. A method for producing amino acids by a fermentation method, which comprises accumulating amino acids and collecting the amino acids from the culture solution. (2) The method for producing an amino acid according to claim 1, wherein the nutrient medium contains L-aspartic acid or a salt thereof. (3) Amino acid-producing microorganisms are Corynepacterium ha.
Amino acids produced by the genus Previva Thylium or Microbacterium include L-lysine, L-threonine, and L-threonine.
- The method for producing the amino acid according to claim 1 or 2, which is isoleucine or L-arginine. (4) Production of the amino acid according to claim 3, wherein the amino acid-producing microorganism is a microorganism belonging to the genus Corynebacterium or Brevibacterium, and the amino acid produced is L-lysine or L-threonine. Law. (5) The method for producing an amino acid according to claim 3, wherein the amino acid-producing microorganism is a microorganism belonging to the genus Corynebacterium, and the amino acid produced is L-inleucine.